Characterization Of MAPEG In Embryonic Stem Cell-derived Hepatic Tissue System And Organogenesis

Part I MAPEG expression in mouse embryonic stem cell-derived hepatic tissue systemObjectiveTo investigate MAPEG expression in mouse embryonic stem (mES) cell-derived hepatic tissue system in vitro, this information helps to understand the exact nature and potial role of MAPEG during liver development in vitro.Methods1. Based on mES cells differentiated technique, the hepatic tissue system derived from the mES cells was established. The gene expression of undifferentiated ES cells marker OCT4 and liver-specific genes (AFP, ALB, HNF3β,CYP7al and G6P) and cardiac-specific gene(Troponin T) as well as endothelial cells marker (PECAM-1) were detected using RT-PCR method; Troponin T-positive cardiomyocytes, albumin-positive hepatocytes and PECAM-1-positive endothelial cells were verified by immunocytochemistry; to confirm albumin production, cultured medium were analyzed using ELISA.2. The mRNA and protein expression of MAPEG family were assessed by RT-PCR, Western blot as well as Immunohistochemistry. GST activity was investigated for the substrates GSH and CDNB. To determine the effect of reactive nitrogen species on embryonic MGST1, samples were treated with ONOO- and MGST1 dimmer was detected by western blot.Results1. Apparent cell morphological changes were observed during the course of differentiation. Hepatocytes appeared as binuclears, and cardiomyocytes which appeared as spontaneously contracting cell clusters were nearby the hepatocytes. OCT4 mRNA decreased within the culture period, while ALB, AFP, CYP7al, HNF3β, G6P, Troponin T and PECAM-1 increased during the course of differentiation. The hepatic tissue system derived from the mES cells was positive staining with anti-albumin, anti-cardiac-troponin T and anti-PECAM-1 by immunocytochemical method. The albumin production gradually increased from day 4 to day 20, and reached a peak level on day 18. It is more efficient than primary hepatocyte.2. Except for MGST3, gene expressions of other five MAPEG members were demonstrated in a developmental-dependent manner in the derived hepatic tissue.3. The protein expression of MGST1 was not detected until differentiating day 14. It gradually increased by maturation of hepatic tissue. LTC4S and mPGES-1 proteins were slightly reduced, while a stable expression of FLAP appeared during the entire course of differentiation. MGST2 and MGST3 failed to express in the derived hepatic tissue, although mRNA of them do existed.4. The microsomes of mES cell-derived hepatic tissue possessed the MGST1-like catalytic activity about 7.65nmol/min/mg. However, MGST1 from the microsome preparation could not form dimmers as usual when exposed to ONOO-, the same as MGST1 from the mouse E18 liver (copulation plug was detected, which defined as E0).Conclusions1. mES cell-derived hepatic tissue posses MAPEG gene expression features. But not all protein expression could be detected, it may due to their multifaceted roles in morphological and functional development of fetal liver, as well as those protein are not structure protein and they can be induce under certain condition.In addition, mES cell-derived hepatic tissue could be a model used for arachidonic acid related inflammatory pathways.2. MGST1 from mES cell-derived hepatic tissue showed very limited activities, and ONOO- could not cause enzyme activation, the same as fetal mouse liver. Those results indicated that the mES cell-derived hepatic tissue might be less able to detoxify exogenous agents than the adult organism, and thus may be sensitive to the potentially toxic effects of a variety of exogenous agents.3. mES cell-derived hepatic tissue expresses hepatocyte and endothelial cell phenotype as well as liver-specific genes and albumin production. It is an appropriate biological assay system for hepatic developmental biology, liver physiology and pathology, liver metabolism, pharmacology and toxicology as well as regenerative medicine such as cell transplantation and tissue engineering. Part II Characterization of MAPEG family in rat organogenesis in vivoObjectiveCharacterization of MAPEG family in rat organogenesis was investigated, in order to understand the exact nature and potential roles of MAPEG during organogenesis in vivo.Methods1. Organs such as brain, heart, lung, liver, kindey were obtained from embryos (E14,16,18 and 20), newborn and mature rat.2. The mRNA expression of MAPEG family was assessed by RT-PCR.3. The proteins expression patterns were detected by western blot and Immunohistochemistry.4. GST activity was investigated for the substrates GSH and CDNB. To determine the effect of reactive nitrogen species on embryonic MGST1, samples were treated with ONOO- and MGST1 dimmer was detected by Western blot. HPLC was used to analyze mPGES-1 and LTC4 synthesis enzymes activities.Results1. Gene expressions of all MAPEG members except for MGST2 in fetal lung were demonstrated with distinct profiles in organogenesis.2. MGST1 protein was gradually declined during brain development, whereas it was increased dramatically in developing liver and lung. Both MGST3 and LTC4S proteins showed a progressive increase in all five organogenesis. FLAP protein was increased obviously in developing kidney, brain and heart. FLAP protein expression in fetal liver and lung were dramatically increased by E16 and E18, respectively, and then intensively reduced at later stages. mPGES-1 was slightly reduced in developing brain and liver, whereas, it was progressively increased in lung and kidney. mPGES-1 protein expression in embryonie heart was increased first and then decreased later. In comparison, for all the five organs, MGST3, LTC4S, FLAP and mPGES-1 protein levels were reduced in adult organs.3. Functionally, microsomal GST activity was progressively elevated during liver development, while it was assessed in terms of steady-state low levels in embryonic brain and lung. In addition, fetal liver MGST1 could be triggered by ONOO-while embryonic lung MGST1 could not.4. LTC4 synthesis enzymes activities were gradually increased in rat organogenesis, except for lung, MGST2 was partly responsible for LTC4 production. In addition, LTC4 synthesis enzymes activities of fetal organs were lower than that of adults.5. mPGES-1 activity was gradually reduced in brain and liver embryonic developemt, while it was increased during heart, lung and kidney organogenesis.Conclusions1. The characterization of MAPEG was organ-specific, developmentally regulated in rat organogenesis in vivo, which suggested their multifaceted roles in morphological and functional development of fetal organs, as well as those protein are not structure protein and they can be induce under certain condition. In addition, fetal organs have material basis for arachidonic acid related inflammatory pathways.2. Rat fetal liver MGST1 could be triggered by ONOO- through a forming dimeric protein, while rat fetal lung and mouse fetal liver MGST1 could not. It indicated that rat fetal liver could pretect fetus from environmental toxicants that they may be exposed to.In addition; modifications of MGST1 were tissue- and species-specific.3. LTC4 synthesis enzymes (MGST3 and LTC4S) expressions and activities were gradually elevated during rat organogenesis. Except for lung, MGST2 was partly responsible for LTC4 production.These results suggested MGST2 and LTC4 may be involved in morphological and functional development of fetal organs.4. mPGES-1 activity showed organ-specific development patterns, which may due to their different roles in morphological and functional development of fetal organs.